Methods for the quantification (quantitation) of nucleic acids are important in many areas of molecular biology and in particular for molecular diagnostics. At the DNA level such methods are used for example to determine the copy numbers of gene sequences amplified in the genome. Further, methods for the quantification of nucleic acids are used in connection with the determination of mRNA quantities since this is usually a measure for the expression of the respective coding gene.
Among the number of different analytical methods that detect and quantify nucleic acids or nucleic acid sequences, Polymerase Chain Reaction (PCR) has become the most powerful and widespread technology, the principles of which are disclosed in U.S. Pat. Nos. 4,683,195 and 4,683,202 (Mullis et al.). However, a typical PCR reaction by itself and correspondingly a typical reverse-transcriptase PCR (RT-PCR) reaction by itself only yields qualitative data, since, after a phase of exponential or progressive amplification, the amount of amplified nucleic acid reaches a plateau, such that the amount of generated reaction product is not proportional to the initial concentration of the target nucleic acid or target nucleic acid sequence, in particular template DNA. However, an end point analysis reveals the presence or absence of a respective starting nucleic acid. This information is, for certain applications, in particular clinical applications, of high value. For other applications including clinical applications, a quantitative measurement is needed, for example to make a proper diagnosis with respect to certain diseases. For example, precise quantitative measurements are needed to diagnose certain infectious diseases, cancers and autoimmune diseases. It may be useful therapeutically to assess the response of a disease to treatment and make prognoses for recovery. Precise quantitative measurement may also help detect false positives, which can occur if there is any contamination of a sample.